Alcohol dehydrogenases are common enzymes in nature and are often characterized as members of either the MDR (medium-chain dehydrogenase/reductase) or SDR (short-chain dehydrogenase/reductase) protein families. Members of the SDR and MDR families appear to have similar activities though they work via different mechanisms and structures. The SDR superfamily comprises isomerases, lyases and oxidoreductases. The enzymes of this family cover a wide range of substrate specificities including steroids, alcohols, and aromatic compounds, however, most family members are known to be NAD+- or NADP+-dependent oxidoreductases. In the combined SDR superfamily, only a single tyrosine residue is strictly conserved and ascribed a critical enzymatic function. Members of the MDR superfamily are often multimeric enzymes associated with 0, 1, or 2 zinc atoms. Substrates of the MDR enzymes are often alcohols and aldehydes. Six different classes of mammalian ADH isoforms are members of the MDR family. In addition to the MDR and SDR families, alcohol dehydrogenases have also been associated with protein families reflecting iron-dependant enzymes, long-chain enzymes, and several types of prokaryotic enzymes with other cofactor requirements.
Most dehydrogenase proteins function as dimers or tetramers and possess at least two domains: the first domain comprising the coenzyme binding site, and the second domain comprising the substrate binding site. This latter domain determines the substrate specificity and contains the amino acids involved in catalysis. ADHs have a variety of substrate specificities, but act primarily on primary or secondary alcohols, hemiacetals, cyclic secondary alcohols, or on the corresponding aldehydes and ketones. The catalytic role of ADH in mammalian ethanol oxidation is well studied. ADH catalyzes the conversion of ethanol to acetaldehyde using NAD+ as a cofactor. Specifically, the coenzyme binds ADH, followed by an interaction with ethanol, the ethanol is subsequently converted to acetaldehyde and the NAD+ is converted to NADH. Members of the mammalian ADH protein family have varying electrophoretic mobilities, Michaelis constants (binding affinities) for ethanol, and sensitivities to pyrazol inhibition. For instance, class I ADHs have low Km values (less than 5 mM) for ethanol oxidation while class II and class IV ADHs have intermediate Km values (about 30 mM). Class III ADH enzymes are not saturable with ethanol and virtually function exclusively as glutathione-dependent formaldehyde dehydrogenases. Allelic variation of the mammalian genes have been identified. The kinetic properties of the resultant variants differ significantly owing to single amino acid substitutions in the coenzyme binding domains of the enzymes.
Alcohol dehydrogenases play fundamental roles in degradative, synthetic, and detoxification pathways and have been implicated in a variety of critical developmental processes and pathophysiological disease states. For instance, allelic variations of ADH2 and ADH3 appear to influence the susceptibility to alcoholism and alcoholic liver cirrhosis in Asians (Thomasson et al. (1991) Am. J. Hum Genet. 48:677-681, Chao et al. (1994) Hepatology 19:360-366, and Higuchi et al. (1995) Am. J. Psychiatry 152:1219-1221). Furthermore, first-pass metabolism is the difference between the quantity of ethanol that reaches the systemic circulation by the intravenous route and the quantity that entered by the oral dose. Several lines of evidence now indicate that first-pass metabolism of alcohol in humans may occur in the liver via the activity of members of the mammalian ADH family (Yin et al. (1999) Enzymology and Molecular Biology of Carbonyl Metabolism 7, Plenum Publishers, New York).
ADHs are also involved in detoxification pathways. For instance, class III ADH is unsaturable by ethanol and mainly functions as a glutathione-dependant formaldehyde dehydrogenase and is therefore important for the elimination of endogenously formed formaldehyde. ADHs are also involved in the metabolism of nitrobenzaldehyde, a dietary carcinogen. It has been suggested that the lack of σ-ADH in Japanese patients may lead to a decreased detoxification of the dietary carcinogen nitrobenzaldehyde and may possible be linked to the high rate of gastric cancer in Japanese (Baron et al. (1991) Life Sci 49:1929-34; Grab et al. (1977) Cancer Res 37:4181-90 and Seedcake et al. (1980) Rev Ed 9:346-51). ADH is also involved in the activation of 1,2 dimethylhydrazine, an experimentally used procarcinogen.
Retinoic acid is a ligand controlling a nuclear receptor signaling pathway that plays a key role in the regulation of embryonic development, spermatogenesis, and epithelial differentiation (Chambon et al. (1996) FASEB J. 10:940-954 and Mangelsdorf et al. (1995) Cell 83:841-850). The synthesis of retinoic acid occurs via the oxidation of retinol to retinal followed by the conversion of retinal to retinoic acid. Members of the alcohol dehydrogenase and short-chain dehydrogenase/reductase families catalyze the reversible, rate limiting conversion of retinol to retinal, while the oxidation of retinal to retinoic acid is catalyzed by members of the aldehyde dehydrogenase or P450 enzyme families (Deuster et al. (1996) Biochemistry 35:12221-12227). Therefore, members of the ADH family influence the growth and developmental processes mediated by the active metabolite retinoic acid.
ADH metabolism of retinol to retinal is inhibited by ethanol, and this may lead to altered epithelial cell differentiation and malignant cell transformation. Furthermore, it has been suggested that the ability of ethanol to inhibit the oxidation of retinol by ADH underlies the pathology of fetal alcohol syndrome, a birth defect characterized by craniofacial, limb, and brain malformations (Duester el al. (1991) Alcohol Clin Exp Res 15:568-572). Retinoic acid also functions to maintain differentiation of epithelial cells and influences spermatogenesis in adult vertebrates (Chambon et al. (1996) FASEB J. 10:940-954). Data suggests that retinoic acid signaling in spermatogenesis and keratinocyte differentiation may be significantly disrupted by ethanol through ADH pathways. It has been proposed that inhibition of retinol metabolism by ethanol may be responsible for the testicular atrophy and spermatogenesis commonly seen in male chronic alcoholics. Furthermore, skin diseases such as psoriasis, have been associated with heavy drinking.
ADH may also play a role in colorectal cancers. During colorectal carcinogenesis, ADH activity is significantly decreased in polyps and further decreased in cancer tissue. (Egerer et al. (1997) Gastroenterology 112:A1260). Furthermore, epidemiological studies have demonstrated that alcohol consumption is a risk factor for development of oral, esophageal, colorectal, and upper gastrointestinal cancers (Blot et al. (1992) Cancer Res 52:2119s-2123s). The role of ADH in cancers of these various tissues may result from the production of acetaldehyde following oxidation of ethanol by ADH, an alteration in retinol metabolism or through the role of ADH in carcinogen metabolism.
Further functional links between disease and the oxidative/reductive actions of various dehydrogenases are being established. For instance, ERAB is a member of the short-chain dehydrogenase/reductase family. Interactions between and Amyloid β peptide and ERAB have been shown to mediate neurotoxicity and apoptosis in neuronal cell lines (Yan et al. (1997) Nature 389:689-693) and thus are being implicated in the pathogenesis of neurodegenerative disorders like Alzheimer's disease (Oppermann et al. (1999) Enzymology and Molecular Biology of Carbonyl Metabolism 7, Plenum Publishers, New York and Oppermann et al. (1999) FEBS Letters 451:238-242).
Accordingly, ADHs are a major target for drug action and development. Therefore, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown ADHs. The present invention advances the state of the art by providing previously unidentified human alcohol dehydrogenases.